In the field of regenerative medicine, either for therapeutic purposes or for plastic surgery, including skin wrinkles treatment, the principle of in situ administration of cell growth factors, mainly platelets or platelet derived growth factors is now of use.
It was already suggested to add such factors to tissue filling or augmentation or regeneration or wound healing implantable preparations, such as hyaluronic acid preparations, collagen preparations, globin and other organic material preparations, ceramics, and synthetic polymers, including polylactide and polylactide glycolide polymers.
The U.S. Pat. No. 6,949,625 discloses an implantable, including injectable tissue filling, augmentation and healing preparation comprising globin being insoluble at physiological pH, preferably homologous, and more preferably autologous globin, which preparations can optionally comprise healing products or cell growth products, or cells.
The U.S. Pat. No. 7,709,017 discloses preparations which are obtainable from soluble globin or globin derivatives, and which can optionally comprise healing products or BMP type growth factors.
The US patent application 20090312239 discloses preparations of globin being insoluble at physiological pH, which can comprise adhesive, filling, augmentation or healing agents.
The US patent application 20120183501 discloses a new healing method comprising the step of administering to the patient, in need thereof, a preparation of globin being insoluble at physiological pH, which preparation may further comprise platelets, or PDGF type growth factors derived from the platelets of platelet rich plasma (PRP), or recombinant products, or cell growth factors (EGF, FGF, . . . ).
In these preparations, the globin component, being insoluble at physiological pH, was prepared by methods such as separating erythrocytes from the whole blood, inducing the hemolysis of the erythrocytes and submitting the hemoglobin to a heme separation step, the erythrocytes being obtained, for example from blood transfusion pouches, or being separated from a donor blood sample, preferably from the patient.
The US patent application 20090074736 discloses a preparation of globin being insoluble at physiological pH, obtained by submitting the whole blood comprising the erythrocytes and the plasma to a depigmentation step in a medium, such as an acetone containing medium, that extracts or dissolves the heme but leaves, in a substantially undissolved state, the globin and the other constituents of proteinic nature submitted to its action, including albumin, alpha- beta- and gamma-globulins, but also blood clotting factors, as well as the platelet factors in the embodiment where the platelets were not initially separated from the blood for further addition to the globin preparation. According to the application 20090074736, all the blood proteins, the platelets and other cells, are submitted to the acetone process which may induce some denaturation of particular proteins and cannot guarantee that they will keep their original structure in the final preparation.
The state of the art in using blood components for tissue regeneration is well summarized in a recent paper published by Jun Araki et al: 2012(1). A copy of the full introduction of this paper, with the included scientific references is here below reproduced:
““The preparation of platelet-rich plasma (PRP) was originally developed as a method to divide red blood cells (RBCs) and plasma from whole blood (WB) in blood transfusion hematology(2). PRP was first used for hemostasis during surgical operations and platelet transfusions for some thrombocytopenic disorders(3). It was revealed through a number of studies that various bioactive substances including platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-b) and epidermal growth factor (EGF), were discharged from the alpha granules of platelets into plasma when platelets were destroyed and activated(4-5). These growth factors are contained in wound exudates from injured subcutaneous tissue and are important in the early phase of the wound healing process(6). Specifically, they promote stromal stem cell proliferation and angiogenesis and are regarded as key signals in tissue repair/regeneration(7-9).
Autologous PRP has less safety concerns than cell-based regenerative therapies. As such, PRP is an issue of extensive research for tissue engineering and regenerative medicine(10-11). PRP has been therapeutically used to accelerate wound healing and tissue repair in dentistry since 1998(12), and the clinical application of PRP was recently expanded to other fields, including cardiac surgery(13), ophthalmology(14), oral and maxillofacial surgery(15), orthopedic surgery(16), plastic surgery(17-18), sports medicine(19) and cosmetic medicine(20-21). For therapeutic purposes, PRP extracted from WB through a single centrifugation is frequently concentrated by a second centrifugation. The first centrifugation is slow to avoid spinning down the platelets, whereas the second spin is fast, so the platelets are spun down. In this study, we refer to this concentrated PRP by the second spin as platelet-concentrated plasma (PCP) to differentiate it from PRP.
Despite the increasing use of PRP or PCP therapeutically, its reported clinical effects are quite variable(22). In fact, there is even a report that concludes that it is invalid(23). Although many commercialized devices are now available for the clinical preparation of PRP/PCP, there is no standardized protocol for PRP/PCP preparation. Moreover, there have been surprisingly few scientific studies on how to optimize the preparation of human PRP/PCP. For example, there has not been a comprehensive study on how the centrifugal force exerted on WB contained in common laboratory ware affects PRP/PCP yield. There are many reasons for a lack of such studies besides the obvious difficulty of obtaining sufficient quantities of fresh human WB samples. One of the many reasons is the complexity of blood coagulation (fibrin polymerisation) and platelet aggregation. Since the precise and consistent control of fibrinogen and platelets is very difficult, well-designed studies are hard to plan and implement. Another reason is that the evaluation methods for the PRP/PCP products(24-25) have not been consistent among previous studies. Finally blood components and physical properties vary between patient samples, making standardization difficult.””
This introduction ends with the description of the goal of the authors which is to publish a better standardized centrifugation process to prepare PRP and PCP. This technique is complex, time consuming and must be carried out by the doctor or his assistant. At the end of the process, there is no guarantee that PRP will remain sterile, unless working in a specific environment which is rarely available to the doctor or his assistant. Because of the presence of platelets, a final sterilization of PRP through filtration membranes is not possible.
A. P. Sclafani, S. A Mc Cormick: 2011(26) introduce their paper with the following statement:
““Since the time of Pare, modern surgical care has relied on optimization of local tissue conditions to allow wounds to heal unimpeded. With an improved understanding of the effects of local growth factors, surgeons have begun to manipulate the wound environment to promote more rapid and effective healing. Isolated growth factors have been applied with some success topically (becaplermin for diabetic foot ulcers and palifermin for radiation induced mucositis), but platelet-rich plasma (PRP) has been promoted in the last decade as a more natural and more potent method of manipulating wound healing. However the process can be time consuming and the results equivocal(27-28).””
Then they describe their experience with PRFM (platelet-rich fibrin matrix). PRFM must be prepared from PRP by the surgeon or his assistant with a kit, under the trade name “Selphyl”. A sterile manipulation from blood sampling to the final injection is facilitated by the kit, but requires rigorous attention. It takes half an hour. In the clinical use of PRP, fibrinogen in the PRP frequently leads to uncontrollable coagulation and platelet activation. Although fibrin gel may be useful as a controlled-release carrier of growth factors(29) or as a filler injectable(30), the formed fibrin gel is quickly degraded by the physiological fibrinolysis caused by tissue plasminogen activators, at the origin of the release of plasmin being responsible for the enzymatic cleavage of the fibrin network. The globin implant is not sensitive to the fibrinolysis reaction and is surprisingly more resistant than any other blood protein to the local biodegradation, probably because of its insoluble character and its very high local concentration. Also, the formed fibrin gel reduces the final product volume, hinders the easy injection of PRP, and is not preferable in many clinical situations. In any case, activated PRP or PRFM must be injected within few minutes to avoid full coagulation of the calcium containing plasma into the syringe, limiting the time for injection or preventing injection even through large needles. Different kits are commercially available and require the same obligations.
The reference papers cited above by Jun Araki et al: (2012) and A. P. Sclafani, S. A Mc Cormick: (2011) are believed to be representative of the state of the art for tissue regeneration. All describe PRP or derived products for tissue regeneration as the only used blood component for practical use. PRP is a liquid and before its injection it must be “activated” by addition of thrombin and/or calcium cations (Ca++) to generate a solid gel, in order to stick on the wound or/and delay the local diffusion of soluble growth factors and other active soluble components. This solidification of PRP is made possible by activation of fibrinogen and other coagulating factors present in plasma or PRP by thrombin and/or Ca++ addition.